DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
2. Applicant’s election without traverse of Group II, claims 17-23, in the reply filed on 13 July 2026 is acknowledged.
Claim Objections
3. Claim 21 is objected to because of the following informalities: This claim recites the phrase “…comprise protospacer…” it is believed that the phrase should read “…comprise a protospacer…” or equivalent. Appropriate correction is required.
Claim Rejections - 35 USC § 112
4. The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
5. Claims 17-23 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A. Claim 17 recites the phrases “…the spacer oligonucleotide…” and “…the scaffold oligonucleotide…” in lines 11 and 12. There is insufficient antecedent basis for these terms in the claim.
B. Claim 17 recites the phrase “…wherein each oligonucleotide molecule comprises a set of two orthogonal primer sequences…” It is unclear how this is intended to limit these claims, as written. Are the claimed oligonucleotides the primers themselves (i.e., primer sequences) or do they merely comprise orthogonal primer binding sites? The structure of this limitation, and how it affects the metes and bounds of the claim, is unclear and therefore the claim is indefinite.
C. Claims 18-23 are rejected for being dependent on a previously rejected claim.
Claim Rejections - 35 USC § 103
6. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
7. This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
8. Claims 17, 20, 21 and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Lane et al (United States Patent Application No. US 20180273935, published 27 September 2018) in view of Yates et al (United States Patent Application No. US 20200255823, published 13 August 2020) and Leuschner et al (United States Patent Application No. US 20200115728, published 16 April 2020).
Regarding claim 1, Lane teaches a method of preparing a library of single guide RNA molecules (abstract) comprising: a library of DNA cleavage products (i.e., a plurality of DNA oligonucleotides; [0202]) comprising a first adapter having a recognition site for a type II DNA endonuclease ([0202] and [0206]), a second adapter having a T7 promoter ([0226], [0227], FIG 8A), and a sequence corresponding to genomic targeting sites for the guide RNA (i.e., a spacer sequence; [0283]). Regarding the limitation wherein the DNA oligonucleotide molecules comprise two orthogonal primer sequences, this limitation is indefinite as discussed above. In so far as this limitation is intended to establish that the double stranded DNA oligonucleotides comprise orthogonal primer binding sites, it is noted that without claim limitations specifically disclosing the primer and its structure this language is being interpreted as intended use and without further limitation to the claims any sequences in the double stranded DNA oligonucleotide are considered “orthogonal primer sequences.” Lane additionally teaches a region of the double stranded DNA oligonucleotide that, when cleaved by a type II restriction endonuclease, is exploited to ligate an sgRNA constant region to the library of DNA oligonucleotide molecules ([0283]). Without additional claim limitations regarding the structure of the “scaffold overhang sequence,” the region of DNA comprising the type II cleavage site taught by Lane is considered to read on this limitation. It is further noted that the T7 sequence present on the double stranded DNA as taught by Lane comprises the nucleotide sequence “TAG,” i.e., the amber stop codon sequence (FIG 9A). Therefore, the double stranded DNA taught by Lane comprises a stop codon.
Lane further teaches the ligation of an sgRNA constant region (i.e., a plurality of double stranded scaffold fragment sequences having a 5' end) to the plurality of double stranded substrate fragments ([0283]) to generate an sgRNA library (i.e., single guide RNA DNA template molecules; [0282] and [0283]). Lane teaches transcribing the single guide RNA DNA template molecules into a plurality of single guide RNA molecules ([0257] and [0343]).
Lane does not teach that the DNA oligonucleotide, scaffold fragment, type II restriction enzyme, and a ligase are incubated in the same reaction mixture.
However, Yates teaches a similar method of preparing guide strand libraries (abstract) wherein cleavage by a type II restriction enzyme is performed in the same reaction as the ligation reaction ([0282]).
Neither Lane nor Yates teach a type II restriction enzyme that produces a 5' overhang on the double stranded oligonucleotide and the scaffold fragments.
However, Leuschner teaches type II restriction enzymes that produce 5' overhangs (e.g., BsaI) as direct alternates to the type II restriction enzymes used in the single guide RNA DNA library constructure taught by Lane (BsaXI and MmeI; [0115]).
It would have been obvious to one having ordinary skill in the art to have modified the single guide RNA DNA library construction method taught by Lane to have performed the restriction digestion and ligation in the same reaction mixture as taught by Yates, and further to have used a type II restriction enzyme producing a 5' overhang as taught by Leuschner, to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make the modifications taught by Yates because performing the restriction digestion and ligation in the same reaction would save time and reduce material lost in the purification step taught by Lane between restriction digestion and sgRNA body ligation ([0332] – [0334]). The ordinary artisan would have been motivated to make the modification taught by Leuschner because Leuschner teaches a small number of alternative type II restriction enzymes and it would have been obvious for the ordinary artisan to try each of the limited options while testing reaction conditions. In addition, the ordinary artisan would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the methods related to the cleavage and ligation of double stranded DNA molecules.
Regarding claim 20, Lane teaches that the targeting sequence (i.e., the spacer sequence) is 20 nucleotides ([0161]).
Regarding claim 21, Lane teaches that the type II recognition sequences are in the DNA adapters (i.e., not in the targeting/spacer sequence; [0223]) and that the targeting nucleotides are the 20 nucleotides 5' adjacent to a PAM motif (i.e., the targeting/spacer sequence does not comprise the PAM sequence, it is adjacent to it; [0256])
Regarding claim 23, Lane teaches a library of 1,276 guides (i.e., spacer sequences that target more than 103 different nucleic acid molecules; [0257]).
9. Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Lane et al (United States Patent Application No. US 20180273935, published 27 September 2018) in view of Yates et al (United States Patent Application No. US 20200255823, published 13 August 2020) and Leuschner et al (United States Patent Application No. US 20200115728, published 16 April 2020) as applied to claim 17 above, and further in view of Yates et al (A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries, Nucleic Acids Research, 49, 22, published 16 December 2021; hereinafter “Yates2”).
Regarding claim 18, the method of claim 17 is discussed fully above and incorporated here. Lane teaches that the double stranded DNA oligonucleotide molecules are prepared by a series of digestion and ligation reactions from a genomic DNA sample (FIG 8A). Neither Lane, Yates nor Leuschner teach that the double stranded DNA oligonucleotide molecules are prepared from a single stranded DNA oligonucleotide template by primer extension.
However, Yates2 teaches a method of preparing sgRNAs by annealing and extending two single stranded DNA strands (pg. 2 column 1 ¶ 2 and Table S1).
It would have been obvious to one having ordinary skill in the art to have substituted the method of producing the double stranded DNA oligonucleotide molecules taught by Lane (i.e., digestion/ligation) with the annealing/extension method taught by Yates2 to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution in order to reduce the number of steps and streamline the preparation of the double stranded DNA oligonucleotide molecules (for example, ordering a library of single stranded DNAs comprising the spacer sequences and extending them compared to: digesting genomic DNA, ligating a first adapter, digesting the products, ligating a second adapter as taught by Lane). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the production of CRISPR/Cas9 guide RNAs.
10. Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Lane et al (United States Patent Application No. US 20180273935, published 27 September 2018) in view of Yates et al (United States Patent Application No. US 20200255823, published 13 August 2020) and Leuschner et al (United States Patent Application No. US 20200115728, published 16 April 2020) as applied to claim 17 above, and further in view of Church et al (United States Patent Application No. US 20180320226, published 08 November 2018).
Regarding claim 19, the method of claim 17 is discussed fully above and incorporated here. Lane teaches that the double stranded DNA oligonucleotide molecules are prepared by a series of digestion and ligation reactions from a genomic DNA sample (FIG 8A). Neither Lane, Yates nor Leuschner teach that the double stranded DNA oligonucleotide molecules are prepared from a single stranded DNA oligonucleotide template by PCR amplification.
However, Church teaches a method of preparing sgRNAs by PCR amplification ([0237] and FIG 11).
It would have been obvious to one having ordinary skill in the art to have substituted the method of producing the double stranded DNA oligonucleotide molecules taught by Lane (i.e., digestion/ligation) with the annealing/extension method taught by Church to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution in order to reduce the number of steps and streamline the preparation of the double stranded DNA oligonucleotide molecules (for example, ordering a library of single stranded DNAs comprising the spacer sequences and PCR amplifying them compared to: digesting genomic DNA, ligating a first adapter, digesting the products, ligating a second adapter as taught by Lane). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the production of CRISPR/Cas9 guide RNAs.
11. Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Lane et al (United States Patent Application No. US 20180273935, published 27 September 2018) in view of Yates et al (United States Patent Application No. US 20200255823, published 13 August 2020) and Leuschner et al (United States Patent Application No. US 20200115728, published 16 April 2020) as applied to claim 17 above, and further in view of Levitzki et al (International Patent Application No. WO 2019170899, published 12 September 2019).
Regarding claim 22, the method of claim 17 is discussed fully above and incorporated here. Lane teaches a scaffold fragment to oligonucleotide sequence ratio of 3.5:1 ([0334]). Neither Lane, Yates nor Leuschner teach a scaffold fragment to oligonucleotide sequence ratio of 2:1.
However, Levitzki teaches a 2:1 fragment ratio in golden gate assembly (a DNA assembly method using Type II restriction enzymes and ligases to link DNA fragments in the same reaction vessel; pg. 29, ¶ 2).
It would have been obvious to one having ordinary skill in the art to have substituted the molar ratio of scaffold to oligonucleotide sequences taught by Lane (i.e., 3.5:1) with the molar ratio taught by Levitzki to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution because the prior art teaches a finite number of molar ratios used in golden gate style DNA fragment assembly (e.g., equimolar, 2:1, or ~3:1 as taught by Lane and Yates). Based on the limited number of options provided in the prior art, it would have been obvious to one having ordinary skill in the art to have tried each of them during routine experimentation. In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the digestion of DNA molecules by type II restriction enzymes and their joining by ligation in the same reaction (i.e., Golden Gate assembly).
Conclusion
12. No claims are allowed.
13. Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN ELLIS YOUNG whose telephone number is (703)756-5397. The examiner can normally be reached M-T 0800 - 1630.
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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
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